Radioimmunoassays make up a significant percentage of the immunoassays used in clinical laboratories. Nonradioactive, immunometric or "sandwich" assays using enzymes for signal generation (EIAs) have generally replaced radioimmunoassays for high molecular weight analytes. Low molecular weight analytes such as the steroid hormones, however, are measured primarily by competitive immunoassays using 125I-labeled haptens as the tracer. SeaLite Sciences manufactures and sells AquaLiteTM, a recombinant form of the calcium-activated photoprotein aequorin, which has been conjugated to monoclonal antibodies and used as the signal in sensitive bioluminescent immunometric assays for TSH and Interleukin-6. We propose to synthesize an AquaLite-labeled testosterone for use in a competitive immunoassay format for this steroid hormone. The validation of this bioluminescent assay during the first phase of this project will define the feasibility for use of AquaLite as a general replacement of 125I in competitive immunoassays. The assay will be optimized and compared to currently available assays. Once feasibility is established for the testosterone assay, Phase II of this project will be menu expansion by conversion of additional steroid hormone radioimmunoassays to bioluminescent immunoassays and the development of an FDA approved clinical diagnostic product line for sale by SeaLite Sciences, Inc.